Integrative analysis reveals the potential prognostic roles and immunological values of unc-5 netrin receptor A (UNC5A) in glioma

Background UNC5A had been reported to play crucial roles in multiple cancers. However, little was known about the associations among UNC5A and glioma. Therefore, we first combined scRNA-seq, proteomics, as well as bulk RNA-seq in order to investigate UNC5A’s functions in gliomas. Methods Online databases provided scRNA-seq, proteomics, as well as bulk RNA-seq data on UNC5A in gliomas. The following procedures were conducted in order: QRT-PCR, Norman chart, gene set enrichment analysis (GSEA), and univariate/multifactor Cox regression analyses. We further explored the associations among UNC5A and tumor immunity. Results By comparing gliomas with normal tissues, the TCGA dataset showed a significantly reduced expression of UNC5A, which was also confirmed by GSE50161, GSE4290, and QRT-PCR findings (p < 0.05). In both the TCGA and CGGA datasets, gliomas patients with low-UNC5A expression would have poorer overall survival (OS) prognoses (p < 0.05). ScRNA-seq analysis by the CancerSEA online website presented that UNC5A had a low expression in various glioma clusters and significantly associated with six functional states. Moreover, UNC5A might be a reliable independent biomarker of OS in gliomas patients (p < 0.05). Based on the results of GSEA, UNC5A might be connected to three significant pathways in gliomas. We also successfully created a Norman chart to assess the OS prognoses of these patients. Additionally, in aspects of tumor immunity, the infiltration levels of immune cells in LGG, the immune cell pathways, tumor immune microenvironment, as well as immune checkpoints in both LGG and GBM were revealed to be significantly influenced by UNC5A (p < 0.05). Conclusions UNC5A was found to have prognostic and immunological significance in gliomas, offering patients with gliomas new treatment options.


Introduction
As a primary brain tumor that is highly recurrent or lethal, gliomas are most likely to develop in adults.They are also prone to resistance to apoptosis, genomic instability, invasion, and migration, all of which contribute to a poor prognosis for the disease and have a detrimental emotional and daily impact on patients [1].The World Health Organization categorized

Univariate/multivariate analysis, Norman chart construction and Gene set enrichment analysis
According to the expression of UNC5A and several clinicopathological factors, univariate and multivariate Cox risk analyses were conducted to identify independent biofactors for overall survival (OS) in gliomas, with a threshold of P value below 0.05 [17], and the Norman chart was also constructed to assess the OS prognosis in glioma patients, evaluating it by calibration plots, C-index, and ROC curves using the R "rms" package [18].Gene set enrichment analysis (GSEA) was also performed to seek UNC5A significantly enriched pathways, with the threshold of nominal P values < 0.05 and |Normalized Enrichment Score (NES)|> 1.5 [19].

Relationships among UNC5A and tumor immunity, tumor neoantigen load (TNB), tumor mutational load (TMB), microsatellite instability (MSI) in gliomas
As described in previous articles [20,21], we used Spearman's approach to examine the associations between UNC5A expression and MSI, TMB, and TNB in LGG or GBM via the Sangerbox online platform (http:// www.sange rbox.com/ tool), with P values < 0.05.Meanwhile, UNC5A's associations with the tumor immune microenvironment, checkpoints, and immune cell pathways in LGG or GBM were also evaluated by this online tool [22].The TIMER database (https:// cistr ome.shiny apps.io/ timer/) online website was also applied by us to assess UNC5A's associations with immune cell infiltration levels in both LGG and GBM [23].

UNC5A's mRNA expression levels and OS prognosis in gliomas
By TCGA pan-cancer analysis, we observed that UNC5A's mRNA expression levels were differentially expressed in various cancers, containing glioma samples of LGG and GBM (Fig. 1A).According to the human body heatmap in Fig. 1B, it was evident that UNC5A was less abundant in glioma tissues than in normal samples.In the GSE4290, GSE50161, and TCGA datasets, our results confirmed that UNC5A was lowly expressed in glioma tissues compared with normal tissues, respectively (Fig. 1C-E).Furthermore, UNC5A mRNA levels were substantially reduced in glioma U251 cells, as demonstrated by QRT-PCR data (Fig. 1F).UNC5A's survival analyses in the TCGA and CGGA datasets both showed that high-UNC5A-expressed groups would have better OS prognoses than those in low-UNC5A-expressed groups (Fig. 1G,  H).ROC curves indicated a moderate diagnosis power of UNC5A in gliomas with AUC values of 0.714 (Fig. 1I).

UNC5A's scRNA-seq data in gliomas
ScRNA-seq data of UNC5A in gliomas was conducted by the CancerSEA online website to seek its associations with 14 kinds of functional states, such as angiogenesis, cell cycle, DNA damage, EMT, inflammation, quiescence, apoptosis, differentiation, DNA repair, hypoxia, invasion, proliferation, metastasis, and stemness, as displayed by the bubble chart (Fig. 2A).The TSNE plot showed that UNC5A was lowly expressed in various glioma clusters (Fig. 2B).Besides, UNC5A was found to be significantly associated with invasion, DNA repair, stemness, DNA damage, cell cycle, and metastasis functional states (Fig. 2C).

UNC5A's prognostic roles in gliomas by univariate/multivariate analysis and the Norman chart
According to univariate analysis, chemotherapy status, age, grade, IDH mutation status, PRS type, 1p19q codeletion status, UNC5A expression, and histology were all significantly associated with OS in gliomas (all p < 0.05; Fig. 5A and Table 1).Based on multivariate analysis, grade, age, IDH mutation status, chemotherapy status, UNC5A expression, 1p19q codeletion status, and PRS type were all shown to be independent predictors of OS in glioma patients (all p < 0.05; Fig. 5B and Table 1).In order to give a quantitative way for predicting the 1-, 3-, and 5-year OS rates in glioma patients, we also created a Norman chart incorporating UNC5A expression and clinicopathological characteristics, evaluating it by calibration plots, C-index, and ROC curves (Fig. 5C).Our established Norman chart's AUC value of the ROC curve at one year was 0.858, at three years it was 0.880, at five years it was 0.880, as well as its C-index value was 0.800 (Table 2).In addition, calibration plots indicated a good performance of this Norman chart (Fig. 5D-F).Through the String (https:// string-db.org/) online website, we identified additional proteins, FLRT1, RHOA, RAC1, SRC, CASP3, DCC, NTN1, MAGED1, NEO1, and NTN4, that might be connected to UNC5A and displayed them as PPI networks (Fig. 7A).We used Spearman's approach to examine the associations between UNC5A expression and MSI, TMB, and TNB in LGG or GBM via the Sangerbox online platform (http:// www.sange rbox.com/ tool), with P values < 0.05.However, no significant correlations were revealed (Fig. 7B-D).

UNC5A's relationships with anticancer drug sensitivity
The relationships among UNC5A and antitumor drug sensitivity was investigated via using the CellMiner database, with the threshold of |corFilter|≥ 0.3 and pFilter < 0.01.Finally, eight antitumor drugs significantly associated with UNC5A expression were selected out.Therein, UNC5A was positively correlated with Afatinib, Afp464, E-7820, Entinostat, Lificguat drug sensitivities, indicating patients with low UNC5A expression were more sensitive to these molecular drugs' chemotherapies.UNC5A was negatively correlated with Abiraterone, Carmustine, Ethinyl estradiol drug sensitivities, indicating patients with high UNC5A expression were more sensitive to these molecular drugs' chemotherapies (Fig. 9).

Discussion
Significant advances in the screening, diagnosis, and treatment of glioma have been made in recent years, along with immunotherapy, stereotactic radiation, and novel chemotherapeutic agents being used in the clinic.However, its prognosis remained dismal [24].UNC5A has been reported to play significant roles in the occurrence and progression of various tumors.Nevertheless, not much research has been done on UNC5A in gliomas up to this point.Therefore, in this study, we first combined scRNA-seq, proteomics, and bulk RNA-seq in order to investigate UNC5A's expression, prognosis, associated pathways or drugs, and clinical or immune relevance in gliomas.According to our results, the TCGA dataset showed a significantly reduced expression of UNC5A by comparing gliomas with normal tissues, which was also confirmed by GSE50161, GSE4290, and QRT-PCR findings.In both the TCGA and CGGA datasets, gliomas patients with low-UNC5A expression would have poorer overall survival (OS) prognoses.ScRNA-seq analysis by the CancerSEA online website presented that UNC5A had a low expression in various glioma clusters and significantly associated with six functional states.Moreover, UNC5A might be a reliable independent biomarker of OS in gliomas patients.Based on the results of GSEA, UNC5A might be connected to three significant pathways in gliomas, including the calcium pathway, the Gnrh pathway, and the Jak-stat pathways.We also successfully created a Norman chart to assess the OS prognoses of these patients.Additionally, in aspects of tumor immunity, the infiltration levels of immune cells in LGG, the immune cell pathways, tumor immune microenvironment, as well as immune checkpoints in both LGG and GBM were revealed to be significantly influenced by UNC5A.Additionally, UNC5A was positively correlated with Afatinib, Afp464, E-7820, Entinostat, Lificguat drug sensitivities and In order to acquire an understanding of how UNC5A contributes to the growth of gliomas, GSEA was conducted by us, and three signaling pathways involved with UNC5A in gliomas were identified: the calcium pathway, the GnRh pathway, and the Jak-stat pathway.The calcium signaling pathway was critical to cellular function and directly or indirectly involved in transmembrane cellular transport, thus affecting tumor invasion and metastasis [25].The gonadotropin-releasing hormone (GnRH) signaling pathway controlled reproductive function as well as cancer growth and progression.GnRH regulated the release of various subordinate hormones and had a significant role in regulating the systemic microenvironment https://doi.org/10.1007/s12672-024-01174-yand endocrine regulation.It had been suggested that this pathway might interact with astrocyte [26].Phosphorylation responses were mediated via the JAK/STAT signaling system, which had been linked to the pathophysiology of autoimmune and inflammatory diseases [27,28].Several cytokines that were implicated in the development of inflammatory and autoimmune disorders transduced intracellular signals through the usage of JAK and STAT.Research had demonstrated that neuroinflammatory illnesses such as multiple sclerosis and Parkinson's disease exhibited activation of the JAK/STAT pathway [24].These all demonstrated the possible functions of the three UNC5A-related pathways in gliomas.
In order to better understand the functions that UNC5A played in tumor immunity, we mined the connections among UNC5A and immunological characteristics like TMB, MSI, TNB, tumor microenvironment, immune checkpoints, immune cell infiltrations, and immune cell pathways.According to our results, UNC5A presented significant associations with immune cell infiltrations in LGG and with immune checkpoints, tumor immune microenvironment, as well as immune cells in LGG or GBM.Upon analyzing immune checkpoints, we also discovered that LGG had significantly more significant immune checkpoints than GBM.GBM was shown to be only significantly associated with ADORA2A, CD200, CD44, and CD48.It had to be confirmed whether this suggested a more noteworthy clinical importance for UNC5A in LGG [29][30][31][32][33].It had been shown that ADORA2A (adenosine A2a receptor) expression was associated with glioma development and it was found to be significantly more immunoreactive in gliomas containing infiltrating tumor cells than in normal brain tissue [32].In their study of the effects of chondroitin sulfate on glioma, zhu et al. found that this drug promoted CD44 degradation, inhibited integrin β1 expression in glioma cells and thereby slowed down the aggressiveness of glioma, indicating a non-negligible role of CD44 in glioma [34].Meanwhile, CD48 had also been studied and its high expression in gliomas might lead to a lower survival rate, while for CD200, its higher expression might prolong microglia activation and tumor growth.The same results were obtained in a Truncated form of CD200 (CD200S) study [35,36].The above evidence also indirectly indicated the immune mechanism that UNC5A might affect in glioma.
Finally, we investigated the relationships between UNC5A and oncologic drug sensitivity and identified eight drugs of interest.Therein, afatinib, a drug for non-small cell lung cancer, achieved its therapeutic goal through potent, irreversible dual inhibition of EGFR and HER2 tyrosine kinases [37,38].AFP464, inhibited angiogenesis by suppressing the expression of integrin alpha2 subunits in the endothelium, and also reported to have antitumor effects [39].Entinostat had also demonstrated its antitumor effects through in vitro and animal studies [40].Abiraterone, a mainstream drug in the treatment of prostate cancer, inhibited its development by inhibiting CYP17 and androgen production to achieve a chemical depot effect [41,42].Carmustine, was a common drug for the treatment of brain tumors and intracranial metastases, and Ethinyl estradiol was clinically valuable for the regulation of sex hormones [43].These above three drugs had a negative correlation trend with UNC5A and they were commonly used in clinical practice.So, whether some new possibilities could be obtained through clinical studies to observe the chance of glioma development in users of the above drugs remained to be explored.It was also important to acknowledge a few limitations.First, further in vivo and in vitro experiments were necessary to fully understand UNC5A's activities in gliomas.Second, further clinical information was needed to investigate the relationships between UNC5A and immunological relevance, clinical relevance, and survival prognosis.Finally, in this article, UNC5A was measured by sequencing the glioma tumor biopsy.However, UNC5A measured by blood, urine, etc. had not been evaluated, and these shall be conducted in our subsequent articles.So, it was still too early to judge if UNC5A was a good biomarker for clinical applications.

Conclusions
In conclusion, UNC5A was revealed to be a potential biomarker for glioma patients' OS prognoses, potentially taking part in the progression of glioma via the calcium pathway, the Gnrh pathway, and the JAK-STAT pathway.Moreover, UNC5A exerted significant associations with immunity.All of these indicated the prognostic and immunological significance of UNC5A in gliomas, offering patients with gliomas new treatment options.

Fig. 6
Fig. 6 Signaling pathways of UNC5A that might be involved in glioma by gene set enrichment analysis; A Calcium signaling pathway; B Gnrh signaling pathway; C Jak-stat signaling pathway; D All of three above mentioned signaling pathways

Fig. 7
Fig. 7 Construction of UNC5A PPI network in glioma and its associations with MSI, TMB, and TNB; A PPI networks associated with UNC5A; B Relevance of UNC5A to MSI; C Relevance of UNC5A to TNB; D Relevance of UNC5A to TMB

Fig. 8
Fig. 8 Immunological features of UNC5A in glioma patients; A Associations between UNC5A and immune infiltration in LGG; B Associations between UNC5A and immune infiltration in GBM; C Associations between UNC5A and tumor microenvironment in LGG; D Associations between UNC5A and tumor microenvironment in GBM; E Expression of UNC5A-related immune checkpoint genes in different tumors; F Expression of UNC5A-related immune cells pathways in different tumors.*p < 0.05; **p < 0.01; ***p < 0.001

Table 3
Gene set enrichment analysis (GSEA) results